| Andrographis paniculata(Burm.f.)nees,i’s annual herb medicine of Acanthaceae,which be contained in the 2015 edition of Pharmacopoeia of People’s Republic of China.It exhibits anticancer,antidiabetic,anti-HIV,antibacterial,antiinflammatory,anti-malarial,anti-angiogenic,hepatoprotective and cardioprotective properties.Objective:Due to the genetic background of the germplasm,the ecological origin,cultivation techniques and harvesting time.These factors influence A.paniculata breeding.The strictly closed flowers fertilization method of A.paniculata also cause the reduction.Therefore,it is difficult to choose excellent female parents for hybrid breeding,thus increasing the risk of extinction.These factors induce an urgent need to improve the breeding of A.paniculata.In this report,we chose different chemical mutagens to induce A.paniculata,in order to provide a basis for the breeding of high quality varieties of A.paniculata.Method:In this study,three chemical mutagens(Oryzalin,Ethylmethane sulfonate(EMS)and Colchicine)induced by A.paniculata seeds as experimental material,the establishment of relevant guidance system,on the basis of the application of plant physiology,biochemistry and molecular biology and other disciplines of knowledge.1.A.paniculata seed was studied by three kinds of chemical mutagen induced,comparison of effects of A.paniculata seed germination,root and study them for the budding difference and influence of seeds,root length,seedling height changes.2.To observe the growth of A.paniculata after induced,studying the changes of morphological characteristics in the process of growth and development,and comparing the characteristics of tetraploid and diploid plants characters.3.Depend on chemical related research methods,study the chemical mutation in the growth process of A.paniculata at soluble sugar,soluble protein,chlorophyll,malondialdehyde and activity of peroxidase and catalase changes.4.Using ELISA method to study the changes of endogenous hormones in A.paniculata at different growth stages,including auxin,abscisic acid,gibberellin and cytokinin.5.Using vertical plate polyacrylamide gel electrophoresis method,the changes of POD,PPO and EST Isozyme in different tissues and organs of A.paniculata were studied by chemical mutagenesis.6.The effects of chemical mutagenesis on the main active components of andrographolide and dehydroandrographolide content were studied by HPLC.7.Based on molecular techniques,the relative expression of ApCPS gene of key enzyme in A.paniculata plants induced by different chemical mutagens was studied.Results:The main results of this study are as follows:1.To study their effects on seed germination and growth,found that the chemical mutagen induced root and bud of the three groups of A.paniculata seeds out after prolonged,and the rooting rate and germination rate decreased,root length and seedling height were also affected.While seed is restrained,and the root rate and germination rate correspondingly decreased,root length and seedling height were also affected.Especially the colchicine group,is the lowest rate of only 62.92%,while the germination rate has the lowest at oryzalin group is only 0.50%,and root length and shoot height of the lowest value,the inhibitory effect is the largest.In a word,the chemical mutagen induced growth of seeds is as follows:the control group≈EMS group>colchicine group>ORYZALIN group.2.Study of chemical induced plant growth and development,found many variants,including oryzalin group were observed in 307 strains of mutant,the total frequency of 75.25%,the variation characteristics of leaves turn yellow,leaf color darker,parietal degradation,deformed,and alien and deep variants.A total of 97 strains were observed in the EMS group with a mutation frequency of 43.32%.Including leaf color,leaf spot,leaf curl,alien.Colchicine group were observed in 168 strains of mutant,the mutation frequency is only 25.26%.Including leaf spots,leaf color,leaf shape,heart-shaped leaves and cotyledon degradation,cotyledon curl,long leaves,parietal lobe degeneration,two parietal degradation,three mutations in the form of leaves,leaf margin serrate,etc.And among them,oryzalin induced tetraploid probability is 12.32%,colchicine induced tetraploid probability of 4.31%.And control group plants compared to tetraploid plants which induced by oryzalin and colchicine,the results show that the tetraploid plants more short,stem diameter larger,leaf surface texture obvious thickening,leaf color is more thick,cuttlefish length shorter,bud number more and more fruit.3.Found that the content of soluble sugar is ascend,especially oryzalin group and colchicine group,its content is significantly higher than the other two groups.And soluble protein content decreased firstly and increased subsequently,and oryzalin group and colchicine group is higher than that of EMS group and control group.Found in chlorophyll content determination of EMS group of chlorophyll content is the lowest,and oryzalin group and colchicine group content higher,may be associated with the difference in leaf color during induction.For the determination of the content of MDA,EMS group and control group malondialdehyde content decreased,oryzalin group and colchicine group increased first and then decreased.The activity of POD decreased significantly,the oryzalin group and colchicine group has higher in plants of POD activity.The highest CAT activity in the oryzalin group,and EMS group and control group had no significant difference,the activity is lowest.4.The change of the content of EMS group and colchicine group was significantly higher than the control group in the variation trend at ZR.The content of ABA in the three groups were lower than that of the control group,but the difference between the three groups were not significant.Change characteristics of IAA content found that EMS group and colchicine group IAA content were significantly low,only oryzalin in August there was an obvious peak content.And three induced group of GA3 content in A.paniculata plant influence had little difference,only showed significant changes in the individual month.5.Found that the colchicine group enzyme band number up to 21,EMS group of enzymes with the least number of only 17,while oryzalin group and control group for 20 bands.At the same time,according to the size of the relative mobility,a total of 17 belt of different mobility of enzyme belt,among which 14 oryzalin group of the band,there are 12 band EMS group,15 band colchicine group,control group with 15 of the band,according to the size of the enzyme with relative mobility,the different chemical mutagen have common enzyme band after induction,and different organs of three enzymes in position,or activity is not significant difference on the size,the difference is number and the size of the active band.6.Found that andrographolide and dehydroandrographolide content of tetraploid oryzalin group were 4.44%and 0.10%respectively,andrographolide and dehydroandrographolide content of tetraploid colchicine group were 4.16%and 0.16%,significantly higher than EMS group’s 2.69%,0.02%,and the control group’s 2.12%,0.09%.Namely the tetraploid oryzalin group and tetraploid colchicine group,its amount of total lactones were 5.54%,4.32%,reach the control group content of more than twice of 2.21%.Obviously the effect of A.paniculata plants of tetraploid oryzalin and tetraploid colchicine can significantly improve the content of andrographolide and dehydroandrographolide.7.The relative expression of key enzymes can be seen that tetraploid colchicine group and tetraploid oryzalin group of ApCPS genes at the relative expression quantity is 2.135819,2.747887,more than two times of ApCPS genes relative expression of the control group and EMS group,and this is consistent with the results in front of the liquid phase,because ApCPS gene is the key of diterpene lactone compounds synthesis enzyme.Conclusions:1.The chemical mutagen induced rooting and germination of A.paniculata seeds have been affected to a certain extent,especially by oryzalin induction,germination was inhibited,and after induction,some variations in many plant groups of A.paniculata growth process,and the index of the induced tetraploid forms of A.paniculata were determined.Found the plant was shorter,thicker stem diameter,leaf surface texture thickening,leaf color is more green,flower bud number length is shorter and more,more fruit.2.In the process of chemical mutagen mutagenesis,the physiological metabolism,endogenous hormones and isozyme bands of A.paniculata have different degrees of change,which can be used as a reference for the study of physiological response mechanism.3.The chemical mutagen induced,the main active ingredients of A.paniculata plants(andrographolide and dehydroandrographolide)content and the relative expression of key enzyme genes-ApCPS between the amount is proportional to the change,and the discovery and expression of the volume content of tetraploid plant Andrographis than control plants increased by 2 times,the reference value for ploidy breeding of A.paniculata.Through the study on the basis of different chemical mutagen induced A.paniculata seeds,to observe changes in the process of growth and development after induced mutation from morphological and physiological and biochemical indexes,enzyme activity and two kinds of main lactone components of andrographolide and dehydroandrographolide content,and relative there are significant differences in the expression of the amount of the key enzyme gene ApCPS,changes from three aspects of physiological,biochemical and molecular level to reveal the physiological chemical mutagens on A.paniculata response mechanism,make a new attempt and the relevant theoretical research for chemical mutation breeding,but also provide new research directions and ideas for further development study on the breeding of medicinal plants and related,breeding and good storability increased yield of A.paniculata and lay the theoretical foundation. |
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